Salt overconsumption has been linked to excess morbidity from hypertension. In order to develop solutions to counteract salt overconsumption without sacrificing palatability, it is important to fully understand how salt taste is processed i taste buds. There are at least two pathways in taste buds that encode salt taste. These two pathways are distinguished by their sensitivity to the epithelial sodium channel (ENaC) blocker amiloride. The amiloride-sensitive (AS) pathway is selective for Na+ and Li+ salts and involves epithelial sodium channels (ENaCs). The amiloride-insensitive (AI) pathway is broadly responsive to sodium and non-sodium salts. Although the types of taste cells that express AS and AI salt taste pathways have been proposed, the available data are limited and often inconsistent. Furthermore, it is not clear how selective are salt-responsive cells, and whether their responses to other taste qualities are due to co-expression of different taste receptors in the same taste cell or indirect activation through cell-to-cell communication. In Aim 1 these uncertainties will be resolved by analyzing breadth of tuning and the expression of cell-type marker genes in physiologically identified AS and AI salt taste cells isolated from the fungiform papillae. Fura2 Ca2+ imaging will be used to measure responses to salts (with and without amiloride) and other taste stimuli. Single-cell RT-PCR will be used to test for expression of marker genes for type I, II and III cells, as well as for the expression of channels and receptors that have been implicated in taste transduction Because isolated taste cells should respond only to stimuli for which they express receptors, interpretation of these experiments will not be confounded by the possibility that cell responses to multiple taste qualities are caused by cell-to cell signaling. Thus, the responses of isolated AS and AI salt-responsive taste cells should answer whether salt receptors are ever co-expressed with receptors for other tastes. The influence of the protected microenvironment of the taste bud on salt taste will also be examined by comparing the characteristics of isolated salt-sensitive cells with characteristics of the same population of cells in an intact taste bud. This will be achieved in Aim 2 by comparing salt-responsive PKD2L1-expressing type III cells from PKD2L1-GFP transgenic mice in dissociated and intact taste bud preparations. Tastant-evoked Ca2+ responses will be measured to analyze breadth of tuning, the time-course of taste responses, and the effect of anion on salt taste. These experiments will provide information about the local network interactions used by the taste bud to help encode and accurately transmit taste information. The proposed studies will provide insight into the mechanisms of salt taste. Results from these studies may have significant implications for models of taste coding at the periphery, particularly labeled-line models. Finally, research into cell-to-cell communication in the taste bud may provide insights into the mechanisms underlying practically important perceptual interactions, such as the suppression of bitter by salty taste.